Enhanced Seroconversion to West Nile Virus Proteins in Mice by West Nile Kunjin Replicon Virus-like Particles Expressing Glycoproteins from Crimean–Congo Hemorrhagic Fever Virus

Removal of genes coding for major parts of capsid (C), premembrane (prM), and envelope (E) proteins on the flavivirus genome aborts the production of infectious virus particles where the remaining genome forms a replicon that retains replicability in host cells. The C-prM-E proteins can also be expressed in trans with the flavivirus replicons to generate single-round infectious replicon virus-like particles (RVPs). In this study, we characterized the use of RVPs based on the Kunjin strain of WNV (WNVKUN) as a putative WNV vaccine candidate. In addition, the WNVKUN C-prM-E genes were substituted with the Crimean–Congo hemorrhagic fever virus (CCHFV) genes encoding the glycoproteins Gn and Gc to generate a WNVKUN replicon expressing the CCHFV proteins. To generate RVPs, the WNVKUN replicon was transfected into a cell line expressing the WNVKUN C-prM-E. Using immunoblotting and immunofluorescence assays, we showed that the replicon can express the CCHFV Gn and Gc proteins and the RVPs can transduce cells to express WNVKUN proteins and the CCHFV Gn and Gc proteins. Our study also revealed that these RVPs have potential as a vaccine platform with low risk of recombination as it infects cells only in one cycle. The immunization of mice with the RVPs resulted in high seroconversion to both WNV E and NS1 but limited seroconversion to CCHFV Gn and Gc proteins. Interestingly, we found that there was enhanced production of WNV E, NS1 antibodies, and neutralizing antibodies by the inclusion of CCHFV Gc and Gn into WNVKUN RVPs. Thus, this study indicates a complementary effect of the CCHFV Gn and Gc proteins on the immunogenicity by WNVKUN RVPs, which may be applied to develop a future vaccine against the WNV.


Introduction
The neurotropic West Nile virus (WNV) consists of many lineages, among which the lineage 1 has been associated with the majority of outbreaks [1]. In this group, infections of the New York 1999 strain (WNV NY99 ) can result in severe meningoencephalitis with a fatality rate around 1% [2]. However, there is no approved human vaccine or antiviral treatment for the infection. In the same lineage, infections of the Kunjin strain (WNV KUN ), a naturally attenuated WNV, has resulted in only a few nonfatal disease cases since 1960 [3,4]. Furthermore, WNV NY99 and WNV KUN share 89% of the envelop (E) gene, 88% of the non-structure (NS) 5 gene and the 3' untranslated region (UTR) [3]. Therefore, WNV KUN could be used as a strategy to develop a vaccine against WNV NY99. WNV NY99 and WNV KUN are members of the family Flaviviridae, which are enveloped viruses with icosahedral structures. The viral genome, which is an approximately 11 kb positive-sense single-stranded RNA (+ssRNA), codes for a single polyprotein. Cleavages of the polyproteins by viral and cellular proteases result in three structural proteinscapsid (C), precursor membrane (prM), and E for viral assembly, as well as seven NS proteins. In addition, the viral genomic RNA is flanked by 5 -and 3 -UTRs which are required for translation, replication, and assembly [5][6][7][8].
The removal of the C-prM-E genes in the flavivirus genome results in replicons that can replicate in transfected cells but abolish production of infectious virus particles [9][10][11][12]. WNV KUN was one of the first flavivirus replicon systems constructed [13]. The replicon can be co-expressed with the corresponding structural genes, resulting in the packaging of replicon and the generation of replicon virus-like particles (RVPs). Several studies have demonstrated the potential of flavivirus RVPs as a vaccine platform [14][15][16][17].
Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly virulent virus that has a fatality rate between 5 and 30%, depending on the outbreaks [18]. As with other members of the genus Orthonairovirus and family Nairoviridae, the virus is an enveloped negative-sense RNA virus. The virus has three genomic segments: small, medium, and large encoding nucleoproteins; glycoproteins; and RNA-dependent RNA polymerase, respectively. The virus infects humans mainly through Hyalomma ticks, which live throughout Africa, Southern and Eastern Europe, the Middle East, India, and Asia [19].
As flavivirus RVPs can be used as a vector to transduce cells and express proteins of interest, they are potentially employed as a multiple vaccine platform. Though CCHFV and WNV infect human and livestock by different vectors, i.e., tick and mosquito, respectively, there are overlapping geographic distributions of the two viruses in Western Asia and Balkan Europe [20][21][22][23]. In this study, we generated WNV KUN RVPs that deliver genes coding glycoproteins CCHFV Gn and Gc to infected cells. These RVPs were then administered to mice to examine their potential as a vaccine candidate. The administration of RVPs into mice induced seroconversion, generating antibodies against CCHFV Gn, CCHFV Gc, WNV NY99 NS1, and WNV NY99 E. However, serum from the CCHFV Gn-Gc RVP-injected mice limitedly neutralize CCHFV but enhanced the neutralization of WNV KUN and seroconversion to WNV NY99 NS1 and E. The data in this study highlight a strategy using the WNV KUN RVPs with the CCHFV Gn-Gc as a vaccine against WNV.

CCHFV Gn-Gc Expression by the WNV KUN Replicon
As we aimed to generate mutivalent WNV KUN RVPs that can transduce the CCHFV Gn-Gc gene, we initially generated a DNA WNV KUN replicon that can express the CCHFV glycoproteins Gn and Gc, termed Gn-Gc replicon. Here, the luciferase (Luc) reporter gene from the previously described WNV KUN replicon [24] was substituted with genes encoding CCHFV Gn-Gc with the foot-and-mouth disease virus autoprotease 2a (FMDV2A) gene inserted between Gn and Gc, allowing for the cleavage of the Gn-Gc into Gn and Gc during replicon expression ( Figure 1A). The replicons were then transfected into BHK-21 cells stably expressing WNV KUN C-prM-E, as described previously [24]. Compared to the Luc replicon, the Gn-Gc replicon expressed the CCHFV Gn and Gc protein as expected ( Figure 1B). In addition, both replicons also expressed the WNV KUN NS1 protein ( Figure 1B). All together, these suggest the replicons can express the WNV KUN polyprotein and the inserted CCHFV Gn and Gc genes.

WNV KUN RVPs Transduced Their Polyprotein Gene and CCHFV Gn-Gc Gene in A Single Cycle
As the Gn-Gc replicon could express the Gn and the Gc proteins, we characterized the packaging of the replicon by the C-prM-E proteins to generate RVPs and its safety. We initially transfected the Gn-Gc replicon into the WNV KUN C-prM-E BHK-21 cell line as previously described [24]. Here, CCHFV Gn, Gc, and WNV KUN NS1 could be visualized, indicating replicon expression, and staining of dsRNA revealed replicon replication ( Figure 2).
As the cell line can express the C-prM-E proteins, the replicon can be encapsidated and enveloped to generate RVPs. To characterize this postulation, we collected the supernatant from transfected cells and infected naïve BHK-21 cells in two consecutive cycles. Media from the RVP-generating cells were diluted to the dilutions with the highest numbers of cells infected. In the first round of infection, cells were transduced to express the CCHFV Gn, Gc, and WNV KUN NS1 proteins, and immunostainings revealed replicon replication ( Figure 2). Because naïve BHK-21 cells do not have the gene encoding C-prM-E, the produced replicon should not be able to be packaged to form new RVPs. As expected, BHK-21 infected in the second cycle could not express the replicon. These results indicate that the Gn-Gc replicon can be packaged to generate RVPs and that the RVPs infect cells only in a single cycle, similarly to the Luc RVPs [24].

CCHFV Gn-Gc RVPs Induced Seroconversion to CCHFV Gn and Gc Proteins and Enhanced Seroconversion to WNV NY99 NS1 and E Proteins
As we demonstrated that RVPs could transduce cells in vitro, we examined the in vivo immunogenicity by administering them into mice. Mice were divided into three groups and subcutaneously injected with either Gn-Gc RVPs, Luc RVPs, or PBS ( Figure 3A). During the study, mice from all groups were healthy and showed no significant differences in weight ( Figure 3B).
We then determined the antibody titers in sera. Mice immunized with CCHFV Gn-Gc RVPs showed seroconversions to Gn and Gc proteins with the serum titers reaching 1:250 and 1:50 dilutions, respectively ( Figure 3C,D). As there were seroconversions to Gn and Gc proteins by mice injected with the control Luc RVPs, there might be cross-reactivity of WNV KUN RVPs antibodies to the Gn-Gc protein. Mice were subcutaneously immunized three times at weeks 0, 2, and 5 with RVPs expressing CCHFV Gn-Gc (6 mice), Luc (6 mice), or phosphate-buffered saline (PBS) (3 mice). (B) Mouse weight from one week before the experiment to the mouse-euthanized day. Mice sera from the study groups were diluted and assayed with enzyme-linked immunosorbent assays (ELISA) to measure antibody titers against CCHFV Gn (C), CCHFV Gc (D) WNV NY99 E (E), and WNV NY99 NS1 (F). The end-point titers were determined as there was no difference in the measured optical density values at 450 nm (OD450) between the vaccinated group and the control group. The experiments were conducted with two technical repeats. The p values are indicated using * p < 0.05 and ** p < 0.01. (G) Serum titers that elicited 50% reduction in the WNV KUN plaque number. Sera from experimented animal were combined before assaying.
As the WNV KUN replicon is packaged by C-prM-E to generate RVPs and the WNV KUN is the attenuated strain of WNV NY99 [3,4], we determined the seroconversion of the RVPs to the WNV NY99 E and NS1. Mice immunized with either Gn-Gc RVPs or Luc RVPs showed seroconversion to the WNV NY99 E and NS1, whose antibody titers can reach to more than 1:6250 and 1:25,000 dilutions, respectively ( Figure 3E,F). Interestingly, there was a significant enhancement of seroconversion to these proteins in mice immunized by the Gn-Gc RVPs, compared to the Luc RVPs.
Ultimately, we examined virus neutralization by sera. As WNV KUN share 89% of the envelop (E) gene to WNV NY99 [3] and the WNV KUN virus in a neutralization assay is often employed in the context of WNV NY99 vaccine development [25,26], neutralization to WNV KUN by the sera was examined. As expected, pooled sera from all mice administrated with the Gn-Gc RVPs showed the enhanced effect of neutralizing the WNV KUN , as the antibody titer with a 50% reduction in plaque numbers was 1:250, compared to 1:50 in mice administered with Luc RVPs ( Figure 3G).
To eliminate the effect of cross-reactivity, we examined CCHFV neutralization by the sera from the Gn-Gc RVP group compared to the Luc RVP group. Sera from the Gn-Gc RVPs were divided into two groups based on a high or low level of antibodies before assaying. In accordance with the antibody levels of the two groups, at the first serum dilution (1:8), diluted sera from the mice with a higher Gc antibody level and these from the mice with a lower Gc antibody level showed around 35% and 15% neutralization of infection, respectively, compared to sera from the control (RVPs-Luc) immunized mice (data not shown).
These data suggest that the RVPs can induce seroconversion to antibodies against the CCHFV and WNV NY99 proteins. In addition, the CCHFV Gn-Gc can enhance the seroconversion to the WNV NY99 proteins, suggesting that they can be incorporated to the WNV KUN RVPs, enhancing the potentials of the RVPs as a putative vaccine candidate against WNV.

Discussion
Since the last outbreak in New York 1999, WNV NY99 has caused nearly 25,000 cases with neuronal invasive symptoms (encephalitis and meningitis) and more than 2000 deaths in North America [2]. In Europe, the virus has caused more than 2000 disease cases and 181 deaths [27]. Thus, there is need to have approved vaccines against the virus, preventing future pandemic burden. There have been six vaccine candidates against WNV in the clinical trial phases I and II, including DNA vector expressing WNV NY99 E, recombinant WNV NY99 E protein, inactivated WNV NY99 , inactivated WNV KUN , live chimeric WNV NY99dengue 4 virus, and live chimeric WNV NY99 -yellow fever virus 17D [28,29].
As WNV NY99 neurological disease is more severe in the elderly, the major target of vaccines against the virus should be the immunosenescent population. In this context, the immunogenicity and safety of the vaccine are essential. The WNV KUN RVP platform is based on the WNV KUN virus, which is an attenuated strain of WNV [3,4] . As the particle contains replicons with no viral packaging genes, it can infect cells in a single cycle, abolishing systemic infection. As a strategy for vaccine development, it is a compromise between inactivated and live-attenuated virus vaccines. Unlike inactivated virus particles, WNV KUN RVPs can preferentially infect and transduce antigen-presenting cells, such as macrophages or dendritic cells [30][31][32][33], and express vaccine candidate proteins, therefore inducing a greater immune response. Compared to live-attenuated virus vaccines, the infectivity of RVPs is limited to a single round, which makes them a safer vaccine platform for the immunosenescent population.
In this study, we characterized the expression and the safety of WNV KUN RVPs, which deliver CCHFV Gn and Gc. Without the CCHFV nucleoprotein, these two proteins cannot function as an CCHFV packaging system. Thus, there is no risk for generation of a chimeric WNV KUN -CCHFV virus using the RVP platform. Indeed, we showed that the RVPs with these genes only infected cells in a single round and mice injected with the RVP survived.
Immunization of the RVPs elicited seroconversions to the antibodies in mice. The Gn-Gc RVPs also conferred the production of antibodies against the WNV NY99 NS1 and E, given that WNV KUN is an attenuated strain of WNV NY99 . Interestingly, the expression of CCHFV Gn and Gc enhanced the immunogenicity of the WNV NY99 E, NS1, and WNV KUN neutralization, which requires further investigation. Indeed, many epitopes within the CCHFV Gn and Gc have been predicted to induce an immune response to CD8 + , CD4 + , and linear B cells, which may suggest the enhanced antibody production to WNV NY99 E and NS1 by Gn and Gc [34,35].
Furthermore, the Gn-Gc RVPs from this study could not induce strong neutralizing antibodies against CCHFV. Likewise, the neutralizing antibody response from CCHFV survivors is also usually low, ranging from 1:8 to 1:32 according to fluorescent focus reduction tests [36]. Interestingly, epitope mapping from CCHFV survivors indicated that antibody responses toward epitopes within Gn and Gc are not likely to elicit neutralizing antibodies [37], but passive immunization with these antibodies was able to protect against CCHFV, highlighting the roles of other mechanisms, such as antibody-dependent cell-mediated cytotoxicity. Thus, although mice sera did not strongly induce CCHFV neutralization, future studies should examine the T cell response to the CCHFV by the Gn-Gc RVPs.

Preparation of Gene Constructs
The WNV KUN replicon was constructed based on the WNV KUN sequence (accession number AY274504), as described previously [24]. To generate the WNV KUN replicon expressing the CCHFV Gn and Gc, the CCHFV Hoti strain Gn and Gc genes were PCR-amplified from the M segment cDNA clone (accession number MH483985.1) with suitable primers to create a FMDV2A between the Gn and Gc and suitable restriction sites flanking the Gn-Gc. The fragment was then substituted with the Luc gene in the WNV KUN replicon by suitable restriction reactions and ligations.

RVP Purification and RVP Concentration Measurement
The RVPs were purified as previously described [24]. In short, supernatants from the RVP production system were collected then loaded on 25% sucrose (Sigma), following by ultra-centrifugations. After centrifugation, both the supernatant and the sucrose were removed, and the RVPs were dissolved in DMEM. To monitor RVP concentration, diluted RVPs were utilized to infect confluent BHK-21 cells for 1 h at 37 • C in 5% CO 2 incubator. The cells were immunofluorescent labeled two days after the infection to count the number of infected cells revealing the number of RVPs.

Sample Collection
Three weeks after the last RVP immunization, the mice were anesthetized with isoflurane for collection of retro-orbital blood samples, after which they were euthanized. The blood was incubated at RT for 30 min before centrifugation at 2000× g for 10 min at 20 • C. The sera were collected and stored at −80 • C until use.

Enzyme-Linked Immunosorbent Assays (ELISA)
To detect antibodies against CCHFV Gn, Gc, WNV NY99 NS1, and E in sera, an in-house ELISA was developed using Nunc Maxisorb plates (Invitrogen) coated with the CCHFV Gn, Gc, WNV NY99 NS1, or E proteins at RT overnight. The plates were then blocked with PBS with 1% BSA for 2 h at RT and washed three times with PBST. Next, 100 µL of diluted sera were added to each well and incubated for 1 h at RT on a shaker at 700 rpm. After washing, horseradish peroxidase (HRP)-conjugated anti-mouse antibody (1:10,000) was added and incubated at RT for 1 h. After washing, 100 µL of substrate solution (ABcam), containing tetramethylbenzidine and peroxide, was added. Plates were then incubated for 15 min before adding 100 µL of stopping solution containing 1M of HCl. The absorbance was read at 450 nm using the Cytation 3 Multi-Mode Reader (BioTek, Bad Friedrichshall, Germany).

WNV KUN and CCHFV Neutralization Assays
Sera collected from each group were pooled and heat-inactivated by incubation at 56 • C for 30 min before being 5-fold serially diluted in DMEM. WNV KUN was rescued from infectious clones, as previously described [38], and was diluted so that there were approximately 50 infectious particles per volume. Next, the diluted sera were incubated for 1 h at 37 • C before infecting 90% confluent Vero cells growing on 24 well-plates for 1 h at 37 • C in 5% CO 2 . After the infection, cells were overlaid with DMEM supplemented with 1.2% Avicel (FMC, Philadelphia, PA, USA), 2% HI-FBS, 1X nonessential amino acids (Gibco), and 1% PEST. After 3 days, the overlays were removed, and cells were fixed by methanol (Fisher, Trinidad and Tobago) for 20 min. The fixed cells were stained with staining buffer containing 2% crystal violet (Sigma), 20% methanol, and 0.1% ammonium oxalate (Sigma) solution for 1 h before washing in water.
The titer of CCHFV neutralizing antibodies in the serum of immunized mice was determined by microneutralization assay. The CCHFV strain Kosova Hoti (07v-EVA70) was obtained from the European Virus Archive. Sera from all mice in the Luc RVPs or the PBS control group were pooled, and sera from mice immunized with CCHFV Gn-Gc RVPs were divided into two groups (high or low anti-CCHFV Gn-Gc antibodies). Thereafter, the sera were heat-inactivated for 30 min at 56 • C. Serial 2-fold dilutions of sera were mixed with 200 CCHFV Hoti viral particles and incubated at 37 • C for 1 h. Thereafter, 100 µL of the serum-virus mix was added in duplicate to Vero cells on a 96-well plate (20,000 cells/well). After 1 h incubation at 37 • C, the inoculum was removed, and cells were washed 3 times with DMEM supplemented with 2% FBS. Then, 100 µL of DMEM supplemented with 2% FBS was added, and the cells were incubated for 24 h at 37 • C and 5% CO 2 . Cells were fixed with an ice-cold acetone-methanol mix (1:1) in −20 • C overnight and stained for CCHFV nucleoprotein for enumeration of fluorescent foci. Total numbers of infected cells in each well were counted and the result was expressed as percent neutralization compared to infection in wells with serum from the control group for each dilution.

Statistics
Statistical differences between the means of parametric data (weights) were determined using two-way ANOVA, followed by Bonferroni correction, while statistical differences between non-parametric data (absorbance values) were determined using Mann-Whitney test. GraphPad Prism 9 was used to perform all statistical analyses. The values are presented as mean ± standard error of the mean.

Conclusions
In conclusion, this study has shed new light on ways to improve the WNV KUN RVPs as a vaccine platform against WNV. Our results suggest that the WNV KUN RVPs have the capacity to transduce cells to express CCHFV Gn and Gc. The RVPs also enhanced the seroconversion and the production of neutralizing antibodies against WNV, which can increase the potential of the RVPs as a vaccine strategy against WNV.

Data Availability Statement:
The data presented in this study are available in this article. Remaining data supporting reported results are available from the corresponding authors upon reasonable requests.

Conflicts of Interest:
The authors declare no conflict of interest.